Effect of VA on the expression levels of p53, p21, cyclin D1 and cyclin E in MCF-7 and MDA-MB-231 cells.

<p>Time-dependent regulation by VA in (A) MCF-7 cells and (C) MDA-MB-231 cells. Cells were treated with VA (50 µg/ml) for 48 h. The data are representative of at least three independent experiments. (B) and (D) Protein quantification of the western blot results shown in (A) and (C) respectively. Protein levels were normalized to the β-actin level and are shown relative to the DMSO-treated control cells (normalized at 1). Statistical differences were analyzed with one-way ANOVA test. **<i>p</i><0.01, *** <i>p</i><0.001.</p

Anti-proliferative effect of VA on human breast cancer cells.

<p>(A) Dose-response curves of VA treatment in MCF-7 and MDA-MB-231 cells. Cells were cultured in 96-well plates and treated with indicated concentrations of VA (0–200 µg/ml) for 24, 48, and 72 h respectively. Cell viability was measured by MTT assay. Data represent the mean ± S.E.M. of three independent experiments. Statistical differences were analyzed with Student's t-test. (B) Comparison of IC₅₀ of VA in MCF-7 and MDA-MB-231 cells at different time points. Values were derived from the graph of growth inhibition against drug concentration (µg/ml) from MTT assay. Data represent the mean ± S.E.M. of three independent experiments.</p

VA induced time-dependent apoptosis in MCF-7 cells.

<p>Cells were treated with VA (50 µg/ml) up to 48 h. Whole-cell lysates were resolved on SDS-PAGE gel and probed with the indicated antibodies. β-actin was used as a loading control. (A) Cleavage of PARP and down-regulation of procaspases were detected by western blot. (C) Down-regulation of anti-apoptotic proteins (Bcl-2 and Bcl-xL) and up-regulation of pro-apoptotic proteins (Bax and Bak) were observed by western blot. The data are representative of at least three independent experiments. (B) and (D) Protein quantification of the western blot results shown in (A) and (C) respectively. Protein levels were normalized to the β-actin level and are shown relative to the DMSO-treated control cells (normalized at 1). Statistical differences were analyzed with one-way ANOVA test. *<i>p</i><0.05, **<i>p</i><0.01, *** <i>p</i><0.001</p

VA inhibited ER-α and the phosphorylation of Akt in MCF-7 and MDA-MB-231 cells.

<p>Cells were treated with VA (50 µg/ml) up to 48 h. Whole cell lysates were resolved on SDS-PAGE gel and probed with the indicated antibodies for western blot analysis. β -actin was used as loading control. VA down-regulated the expression of ER-α, p-Akt and p-GSK3 β in (A) MCF-7 cells and (C) MDA-MB-231 cells. The data are representative of three independent experiments. (B) and (D) Protein quantification of the western blot results shown in (A) and (C) respectively. Protein levels were normalized to the β -actin level and are shown relative to the DMSO-treated control cells (normalized at 1). Statistical differences were analyzed with one-way ANOVA test. *<i>p</i><0.05, *** <i>p</i><0.001</p

VA induced caspase-dependent apoptosis in MCF-7 human breast cancer cells.

<p>General caspase inhibitor, z-VAD-fmk alleviated VA-induced apoptosis. Cells were pre-treated with 20 µM of z-VAD-fmk for 2 h followed by co-incubation with VA for 24 h. (A) Quantitative analysis of apoptotic cells after staining with Annexin V-FITC/PI and analysing by flow cytometry. Values shown are means ± S.E.M. of two independent experiments. *<i>p</i><0.05. (B) Western blot analysis of PARP, caspases, and Bcl-2 in the presence of z-VAD-fmk. Whole cell lysates were resolved on SDS-PAGE gel and subjected to western blot analysis. The data are representative of two independent experiments. (C) Protein quantification of the western blot results shown in (B). Protein levels were normalized to the β -actin level and are shown relative to the DMSO-treated control cells (normalized at 1). Statistical differences were analyzed with one-way ANOVA test. *<i>p</i><0.05, **<i>p</i><0.01</p

VA induced p53-independent G1/S cell cycle arrest and apoptosis in MCF-7 cells.

<p>Cells were pre-treated with 20 µM of pifithrin-α (PFT-α) for 2 h followed by co-incubation with VA for 24 h. (A) Inhibitory effects of PFT-α on cell cycle distribution was analyzed by flow cytometric analysis. Cells were starved for 24 h prior to PFT-α and VA exposure. The data are representative of two independent experiments. (B) Annexin V-FITC/PI assay analysis of the effects of PFT-α on VA-induced apoptosis. Values shown are means ± S.E.M. of two independent experiments. ‘ns’ means non-significant (<i>p</i>>0.05). (C) Western blot analysis of the effects of PFT-α on the expression levels of p53, cell cycle regulators and apoptotic-related proteins. The data are representative of three independent experiments. (D) Protein quantification of the western blot results shown in (C). Protein levels were normalized to the β -actin level and are shown relative to the DMSO-treated control cells (normalized at 1). Statistical differences were analyzed with one-way ANOVA test. *<i>p</i><0.05; ns means non-significant (<i>p</i>>0.05).</p

VA induced time-dependent apoptosis in MDA-MB-231 cells.

<p>Cells were treated with VA (50 µg/ml) up to 48 h. Whole-cell lysates were resolved on SDS-PAGE gel and probed with the indicated antibodies. β-actin was used as a loading control. (A) Cleavage of PARP and down-regulation of procaspases were detected by western blot. (C) Down-regulation of anti-apoptotic proteins (Bcl-2 and Bcl-xL) and up-regulation of pro-apoptotic proteins (Bax and Bak) were observed by western blot. The data are representative of at least three independent experiments. (B) and (D) Protein quantification of the western blot results shown in (A) and (C) respectively. Protein levels were normalized to the β -actin level and are shown relative to the DMSO-treated control cells (normalized at 1). Statistical differences were analyzed with one-way ANOVA test. *<i>p</i><0.05, **<i>p</i><0.01, *** <i>p</i><0.001</p

Changes in cell cycle phase distribution after VA treatment for 24, 48 and 72

<p>Flow cytometric analysis of (A) MCF-7 cells and (B) MDA-MB-231 cells in different phases of the cell cycle according to VA concentrations. Cells were cultured and synchronized by serum-free medium for 24 h prior to VA treatment, and followed by staining with propidium iodide. Values shown are means ± S.E.M. of three independent experiments. Statistical differences were analyzed with one-way ANOVA test. *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001</p

The synergistic effect of VA combined with doxorubicin on the growth of MCF-7 cells and MDA-MB-231 cells.

<p>(A) Growth inhibition rate of doxorubicin alone (0.1–100 µg/ml) or in combination with VA (30 µg/ml) after exposure for 24 h, 48 h and 72 h. Inhibitory effect was determined using MTT assay. (B) Coefficient of drug interaction (CDI) values for the combination treatment of doxorubicin with VA (30 µg/ml) on MCF-7 cells. CDI<1 or <0.7 indicate that the drugs are synergistic or significant synergistic respectively. Values shown for all the experiments are means ± S.E.M. of at least three independent experiments. Statistical differences were analyzed with Student's t-test.</p

Quantitative analysis of VA-induced apoptosis in MCF-7 and MDA-MB-231 breast cancer cells as assessed by Annexin V-FITC staining assay.

<p>Cells were treated with indicated concentrations for 24, 48, and 72-FITC and PI staining. Values shown are means ± S.E.M. of two or three independent experiments. Statistical differences were analyzed with one-way ANOVA test. *p<0.05, **p<0.01.</p